**5. Inhibition of bacterial cell division**

One mechanism used to stop biofilms from growing is to inhibit cell division. Peptides having antimicrobial activity inhibit cytoplasmic proteins that play a big role in cell division and also promote cell growth by penetrating the bacterial cytosol through formation of channels at outer membrane or via a flip-flop of phospholipids (when the cell is ready to divide, then the nuclear membrane melts) [14]. Drosocin, pyrrhocoricin, and apidaecin are proline-rich antimicrobial peptides (AMPs). They can impede the initiation of chromosomal DNA (cDNA) replication by binding with a shock protein of bacteria DnaK [23, 24] or bacterial death [25].

## **6. Biofilm's inhibitors based on nucleotide second messenger molecules**

Nucleotide second messenger molecule cyclic di-GMP (c-di-GMP) is involved in biofilm development and the growth of biofilm can be altered by modifying the c-di-GMP signaling pathway **(Figure 4)** [14, 26]. c-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs). Its mechanism of action is achieved by microbial cells reducing the level of c-di-GMP via phosphodiesterase activation due to nitrosative and starvation conditions [27] leading to biofilm dispersion. However, c-di-GMP has three main mechanisms of biofilm formation regulation:


#### **Figure 4.**

*Regulation of cyclic di-GMP on biofilm formation by inhibiting bacterial motility and increasing EPS production [26].*

of c-di-GMP, YciR begins to apply its PDE function to release the inhibition of YdaM and MlrA and concomitantly, YdaM can activate MlrA to enhance the central curli regulator CsgD, thus prompting the transcription of curli genes and enabling the curli formation [31] (**Figure 4C**). c-di-GMP also regulates another essential component of biofilm matrix called bacteria cellulose [32]. Bacterial cellulose synthase (BcsA), is attached in the inner membrane of the cell and contains a catalytic glycosyltransferase domain and a c-di-GMP-binding PilZ domain in its intracellular part [33] (**Figure 4D**). Glycosyltransferase domain is activated when c-di-GMP binds to the PilZ domain of BcsA which allows the bacterial cell to assemble the nascent polysaccharide with the help of the BcsB/ BcsC/BcsZ complex to form extracellular cellulose [33] (**Figure 4D**).
